Linear chromosomes terminate in specialized nucleoprotein structures called telomeres, which are required for genomic stability and cellular proliferation. Telomeres end in an unusual GT-rich 32 single-strand overhang that requires a special cap to prevent inappropriate recognition by the DNA damage machinery. Furthermore, telomeres are not fully replicated by the canonical DNA replication machinery. Highly proliferating cells overcome this limitation through the action of the replicative enzyme telomerase. The implementation of telomere capping and the regulation of telomerase are critical to cellular survival. This research program combines biochemical and structural strategies to understand how telomere factors perform these activities. Our program is built around a central player in telomere maintenance, the family of proteins that target the 32 single-stranded overhang region of the telomere via specialized sequence-specific single-stranded DNA-binding domains. These telomere end-protection (or TEP) proteins are required for normal cellular proliferation, playing a vital role in telomere maintenance by providing a capping function as well as regulating the action of telomerase at the telomere. The regulation of telomerase action at telomeres by TEP proteins is performed in concert with regulatory components of the telomerase holoenzyme, but the mechanism by which this occurs is largely unknown. In Aim 1, we present a focused research plan to understand how telomerase activity is regulated by these factors in the model system S. cerevisiae. We will obtain mechanistic insights into telomerase regulation using an in vitro reconstituted telomerase assay and direct binding assays. Aim 2 expands on our discovery in the previous funding period of a new DNA-binding domain (DBD) in the TEP protein from a second model organism, S. pombe. Our knowledge of the mechanism of ssDNA recognition by TEP proteins is incomplete and the high-resolution structure of the SpPot1-DBD will help identify the common elements of ssDNA-recognition, as well as the molecular mechanisms that mediate alternate specificity. This integrated research program will provide novel insights into the activity of a biologically critical family of proteins by addressing key questions with high-resolution structural and biochemical tools.